Human Mob-5 (IL-24) receptors and uses thereof

ABSTRACT

The present invention provides the receptors for Mob-5 (IL-24). One of the Mob-5 receptors comprises IL-22R1 and IL-20R2. Another Mob-5 receptor comprises IL-20R1 and IL-22R2. The invention also provides methods of inhibiting the Mob-5 receptor as well as methods of detecting cancer by detecting the presence of the Mob-5 receptor.

This application claims priority to provisional U.S. Patent ApplicationNo. 60/315,684, filed Aug. 29, 2001, which is incorporated herein byreference in its entirety.

This invention was made with government support under Grant CA74067 fromthe National Institutes of Health. The U.S. Government may have certainrights in this invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to the receptors for human Mob-5 (IL-24), and thedetection of Mob-5 receptor expression and/or the presence of the Mob-5receptor as potential markers for the early diagnosis of cancer. Methodsfor inhibiting the interaction between Mob-5 and the Mob-5 receptors arealso provided.

2. Background Art

Mob-5 (IL-24), a gene constitutively activated by oncogenic Ras, encodesa secreted protein that exhibits significant homology to the IL-10family of cytokines (See U.S. Application Ser. No. 60/178,185, which isherein incorporated by reference in its entirety). To provide insightinto the biochemical and biological functions of Mob-5/Mda-7 in normaldevelopment as well as during cell transformation and tumorigenesis, thepresent invention provides the identification and characterization ofits functional cell surface receptor. This invention shows that Mob-5 isnormally expressed in activated PBMCs (peripherial blood mononuclearcells) and its expression is deregulated in cancer tissues. Namely,under normal circumstances, Mob-5 (IL-24) is secreted by T cells andbinds to its receptors on target cells at the site of infection orinjury to promote healing, which involves proliferation. This inventionshows that, in cancer, activation of oncogenes such as ras and the lossof p53 tumor-suppressor genes lead to over expression of both Mob-5 andits receptors in the same cells. Thus, such autocrine loops allow cancercells to stimulate themselves for unregulated cell proliferation. Thepresent invention provides compositions and methods for inhibiting orblocking these autocrine loops, thus inhibiting cancer. Furthermore, thepresent invention provides the surprising discovery that Mob-5 binds toa Mob-5 receptor that consists of a new combination of two previouslyknown class II cytokine receptor subunits, thus providing a strongmolecular basis for cross-talk among the IL-10 family of cytokines.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the differential expression of the IL-24 mRNA in humancolorectal cancer. Total RNA from pair-wise matched cancer tissues andtheir adjacent normal tissues from five patients were analyzed by eitherquantitative RT-PCR (top panel) or regular RT-PCR followed by Southernblot using a IL-24 cDNA probe (middle panel). The image from rRNAs usedfor the analysis was shown as a control for equal sample loading.

FIG. 1B shows the induction of IL-24 by activated PBMCs. Freshlyprepared human PBMCs were treated with PBS, LPS or ConA for 2 and 4hours. The resulting conditioned media were subsequently analyzed bywestern blot analysis which showed that ConA, but not LPS, was able tocause a rapid induction of IL-24.

FIG. 2A shows that conditioned media of 293T and 293T stably transfectedwith the IL-24-His expression vector were analyzed by Western blot usinga IL-24 specific antibody.

FIG. 2B shows the glycosylation of human IL-24. The partially purifiedIL-24-His before and after treating with endoglycanase F (PNGase F) wereanalyzed by Western blot using a monoclonal antibody specific to IL-24.Several bands smaller that the untreated IL-24-His were seen afterdigestion with PNGase F, indicating that IL-24-His is indeedglycosylated.

FIG. 3A illustrates western blot analysis of conditioned mediacontaining alkaline phosphatase (AP) or IL-24-AP, using polyclonalantibody against AP. These media were used to conduct the followingIL-24 receptor binding studies.

FIG. 3B shows the receptor binding analysis for IL-24. Cos-E5 cells ( aclonally purified Cos-1) were transiently transfected with expressionvectors (individually or in pair) encoding the corresponding receptorsubunits as indicated. The cells with and without transfection weresubsequently assessed for their ability to bind IL-24-AP (solid bars)versus AP control (open bars). None of the receptor subunits alone,except IL-20R2, exhibited appreciable IL-24-AP specific binding, whichwas substantially potentiated when either IL-20R1 or IL-22R1 wasco-transfected with IL-20R2.

FIG. 3C illustrates cell surface staining assay for the IL-24 receptorbinding. Cos-E5 cells transfected with either the IL-20R1, IL-22R1 andIL20R2 expression vector alone or in pairs were stained for the cellsurface-bound IL-24-AP activity and viewed under a light microscope (20×magnification) without phase contrast. The result was consistent withthat obtained from the quantitative binding study, in that, cellstransfected with both IL-20 receptor (IL-20R1/IL-20R2) heterodimericreceptor exhibited positive staining by IL-24-AP. While cellstransfected with IL-20R2 alone also showed weak staining for IL-24 AP,neither IL-20R1 nor IL22R1 was able to confer IL-24AP binding to thecells.

FIG. 4A is a northern blot analysis showed that HaCaT humankeratinocytes are positive for IL-22R1/IL-20R2 receptor expression.Unlike HaCaT cells, BHK cells do not express either receptor subunit.

FIG. 4B shows IL-24 dependent STAT activation in HaCaT cells. HaCaTcells were stimulated for 30 minutes with either 293T control medium orIL-24-His conditioned medium, and then processed for gel-shift assay forSTAT activation using a ³²P-labled STAT s=specific probe (GRR).

FIG. 5A shows that IL-24 signals through both heterodimeric receptors ofIL-20 (IL20R1/IL-20R2) and IL22R1/IL20R2 by activating STAT-1 andSTAT-3. BHK cells were transiently transfected with the receptor pairsas indicated. After stimulating the cells with either the control 293Tmedium or 293T/IL24-His medium for 30 min, STAT activation was analyzedby a 6% polyacrylamide gel with nuclear extracted from the cells usingthe ³²P-labled GRR probe. IL-24-His was able to confer specificactivation of STATs through both IL-20R1/IL-20R2 and IL22R1/IL20R2heterodimeric receptors.

FIG. 5B illustrates that IL-24 signals through its receptors byactivating both STAT-1 and B3. BHK cells transiently transfected withIL22R1/IL20R2 expression vectors were stimulated with either the 293Tcontrol medium or IL-24His-293T medium for 30 min. Gel-shift assays werecarried out with nuclear extracts using the 32P-labeled GRR probe in theabsence or presence of antibodies against STAT-1 and STAT-3 asindicated. A 5% polyacrylamide gel was used for better separation ofdifferent forms of STAT-GRR complexes. Both STAT-1 and STAT-3 antibodiescaused either disruption (STAT-1) or super-shift (STAT-3, indicated byarrowhead) of STAT-GRR complexes induced by IL-24-His, respectively.

FIG. 6 shows the inability of IL-24 to inhibit LPS induced TNF-αproduction from PBMCs. The freshly prepared human PBMCs were either mocktreated or treated with 80 ng/mL LPS for 4 hours in the absence andpresence of IL-10 (20 ng/mL), IL-24-His (100 ng/mL) or both cytokines asindicated. TNF-α secreted into the media was quantified by ELISA. UnlikeIL-10, IL-24-His had little inhibitory effect on LPS induced TNF-αproduction by PBMCs.

FIG. 7 shows the induction of IL-24 receptor (IL-20R1/IL-20R2) by Ha-rasoncogene. In these experiments REF cells before and after transformationby oncogenic H-ras and mutant p53 (cell lines T101-4 and A1-5) wereanalyzed by Northern blot for the expression of both IL-20 subunits.

FIG. 8 shows that colon cancer cell line, SW480 is positive for IL-24receptor(s). The SW480 cells were stimulated with either 293T controlmedium or IL-24-His conditioned medium for 30 min. STAT activation wasthen analyzed by a gel-shift assay. The arrowhead indicates IL-24specific STAT activation.

SUMMARY OF THE INVENTION

The present invention provides a composition comprising a complexbetween IL-22R1 and IL-20R2, a composition comprising a complex betweenMob-5 (IL-24) and IL-22R1 and IL-20R2, a composition comprising acomplex between Mob-5 (IL-24) and IL-20R1 and IL-20R2, a compositioncomprising a complex between Mob-5 (IL-24) and IL-22R1, a compositioncomprising a complex between Mob-5 (IL-24) and IL-20R2 and a compositioncomprising a complex between Mob-5 (IL-24) and IL-20R1.

The invention further provides purified antibodies that bind to thecomplexes of the present invention.

Further provided by the present invention is a vector comprising anucleic acid encoding IL-22R1 and IL-20R2, and a vector comprising anucleic acid encoding IL-20R1 and IL-20R2.

A method for inhibiting cancer comprising inhibiting the Mob-5 receptorin a cell so as to inhibit Mob-5 induced cancer.

The invention also provides a method of inhibiting a cellulartransformation phenotype induced by an oncogene whose product functionsupstream of a mob-5 gene product, comprising inhibiting formation of aMob-5 receptor in a cell containing the upstream oncogene so as toinhibit the expression of the cellular transformation phenotype of theupstream oncogene.

Also provided is a method for inhibiting a mob-5 induced cellulartransformation phenotype comprising inhibiting the Mob-5 receptor in acell so as to inhibit the expression of the mob-5 induced cellulartransformation phenotype.

The invention also provides a method of detecting the presence of cancerin a patient comprising: contacting a sample from the patient with anantibody to a Mob-5 receptor; detecting the binding of the antibody withan antigen in the sample, wherein binding of antigen to the antibodyindicates the presence of Mob-5 receptor antigen in the sample andwherein Mob-5 receptor antigen in the sample indicates the presence ofcancer in the patient, thereby detecting the presence of cancer in thepatient.

Further provided is a method of detecting the presence of cancer in apatient comprising: contacting a sample from the patient with a Mob-5receptor antigen; detecting the binding of the antigen with an antibodyin the sample, wherein binding of antigen to the antibody indicates thepresence of Mob-5 receptor antibody in the sample and wherein Mob-5receptor antibody in the sample indicates the presence of cancer in thepatient, thereby detecting the presence of cancer in the patient.

The present invention also provides method of detecting the Mob-5receptor comprising, contacting a cell with a labeled Mob-5, detectingthe binding of the labeled Mob-5 to the cell, wherein binding of thelabeled Mob-5 to the cell indicates the cell is a Mob-5 receptorexpressing cell, thus detecting the Mob-5 receptor.

Also provided is a method of screening for an inhibitor of Mob-5 bindingto the Mob-5 receptor comprising: a) contacting a Mob-5 receptorcontaining cell with a labeled Mob-5 protein and a putative inhibitor;and b) measuring the amount of labeled Mob-5 protein bound to the cells,such that a decrease in labeled Mob-5 protein binding as compared toMob-5 protein binding in cells that were not contacted with theinhibitor, indicates the presence of an inhibitor of the interactionbetween Mob-5 and the Mob-5 receptor.

The present invention further provides a method of screening for aninhibitor of IL-22R1/IL-20R2 dimerization comprising: a) transfecting acell with a plasmid containing a nucleic acid comprising a nucleic acidsequence encoding Il-22R1 and a plasmid comprising a nucleic acidsequence encoding Il-20R2; b) contacting the cell with a putativeinhibitor and; c) measuring dimerization, wherein a decrease indimerization in the cell of step b) as compared to dimerization in acell that was not contacted with the putative inhibitor indicates thepresence of an inhibitor of IL-22R1/IL-20R2 dimerization.

The invention also provides a method of screening for an inhibitor ofIL-20R1/IL-20R2 dimerization comprising: a) transfecting a cell with aplasmid containing a nucleic acid comprising a nucleic acid sequenceencoding Il-20R1 and a plasmid comprising a nucleic acid sequenceencoding Il-20R2; b) contacting the cell with a putative inhibitor and;c) measuring dimerization, wherein a decrease in dimerization in thecell of step b) as compared to dimerization in a cell that was notcontacted with the putative inhibitor indicates the presence of aninhibitor of IL-20R1/IL-20R2 dimerization.

Further provided is a method of detecting Mob-5 activation of a Mob-5receptor comprising: a) contacting a cell with Mob-5; b) measuring STATactivation, wherein an increase in STAT activation in the cell of stepa) as compared to STAT activation in a cell not contacted with Mob-5indicates Mob-5 activation of a Mob-5 receptor.

Also provided is a method of screening for an inhibitor of Mob-5activation comprising: a) contacting a Mob-5 receptor containing cellwith Mob-5 and a putative inhibitor; b) measuring STAT activation,wherein a decrease in STAT activation in the cell of step a) as comparedto a Mob-5 receptor containing cell that was not contacted with theputative inhibitor indicates the presence of an inhibitor of Mob-5activation.

The present invention also provides a method of screening agents foranti-cancer activity comprising; a) administering the agent to a cancercell; b) monitoring the activity of a Mob-5 receptor in the cell,whereby an inhibition of the activity of the Mob-5 receptor indicatesthe agent has anti-cancer activity, thereby screening the agent foranti-cancer activity.

DETAILED DESCRIPTION OF THE INVENTION

The present invention may be understood more readily by reference to thefollowing detailed description of the preferred embodiments of theinvention and the Examples included therein.

Before the present compounds and methods are disclosed and described, itis to be understood that this invention is not limited to specificproteins, specific methods, or specific nucleic acids, as such may, ofcourse, vary. It is also to be understood that the terminology usedherein is for the purpose of describing particular embodiments only andis not intended to be limiting.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to “a nucleic acid” includes multiple copies of the nucleicacid and can also include more than one particular species of nucleicacid molecule.

Mob-5 (Il-24) Receptors

The present invention provides receptors for human Mob-5 or IL-24. Onereceptor is comprised of IL-22R1 and IL-20R2 and another receptor iscomprised of IL-20R1 and IL-20R2. The present invention alsocontemplates receptor subunits, for example, IL-22R1, IL-20R2 andIL-20R1 as well as fragments of the receptor and fragments of thereceptor subunits.

The nucleic acid sequence encoding IL-22R1 (SEQ ID NO: 4) and the aminoacid sequence of IL-22R1 (SEQ ID NO: 5) can be accessed on GenBank viaAccession No. NM_(—)021258. IL-22R1 has also been described in theliterature. (See Kotenko et al. AIdentification of the functionalinterleukin-22 (IL-22) receptor complex: the IL-10R2 chain (IL-10Rbeta)is a common chain of both the IL-10 and IL-22 (IL-10-related Tcell-derived inducible factor, IL-TIF) receptor complexes,@ J. Biol.Chem. 276(4), 2725-2732 (2001) and Xie et al. AInterleukin (IL)-22, aNovel Human Cytokine That Signals through the InterferonReceptor-related Proteins CRF2-4 and IL-22R,@ J. Biol. Chem. 275 (40),31335-31339 (2000)) which references are herein incorporated byreference in their entireties.

The nucleic acid sequence encoding IL-20R1 (SEQ ID NO: 6) and the aminoacid sequence of IL-20R1 (SEQ ID NO: 7) can be accessed on GenBank viaAccession No. AF184971. IL-20R1 has also been described in theliterature. (See U.S. Pat. No. 5,945,511 and Blumberg et al.AInterleukin 20: discovery, receptor identification, and role inepidermal function, @ Cell. 104(1):9-19 (2001)) which references areherein incorporated by reference in their entireties.

The nucleic acid sequence encoding IL-20R2 and the amino acid sequenceof IL-20R2 has been described and published in International PatentPublication WO 99/46379 which reference is herein incorporated byreference in its entirety.

It is understood that, where desired, modification and changes may bemade to the receptors, receptor subunits and receptor subunit fragmentsdescribed herein and still obtain a protein having like or otherwisedesirable characteristics. These modifications include insertions,deletions and point mutations. Such changes may occur in naturalisolates or may be synthetically introduced using site-specificmutagenesis, the procedures for which, such as mis-match polymerasechain reaction (PCR), are well known in the art.

For example, certain amino acids may be substituted for other aminoacids in a receptor without appreciable loss of functional activity ofthe Mob-5 receptor. These substitutions can occur in either subunitcomprising the receptor or in both subunits. Since it is the interactivecapacity and nature of a protein that defines that protein's biologicalfunctional activity, certain amino acid sequence substitutions can bemade in a Mob-5 receptor amino acid sequence (or, of course, theunderlying nucleic acid sequence) and nevertheless obtain a Mob-5receptor protein with like properties. It is thus contemplated thatvarious changes may be made in the sequence of the Mob-5 receptor aminoacid sequence (or underlying nucleic acid sequence) without appreciableloss of biological utility or activity and possibly with an increase insuch utility or activity.

It is also understood that the Mob-5 receptors of this invention mayalso contain conservative substitutions where a naturally occurringamino acid is replaced by one having similar properties and which doesnot alter the function of the polypeptide. Such conservativesubstitutions are well known in the art. Thus, it is understood that,where desired, modifications and changes may be made in the nucleic acidand/or amino acid sequence of the Mob-5 receptor protein of the presentinvention and still obtain a Mob-5 receptor protein having like orotherwise desirable characteristics.

The Mob-5 receptor polypeptides of this invention can be obtained viarecombinant methods where a vector containing a nucleic acid encodingthe polypeptide of interest can be introduced into an expression systemthat is capable of producing the polypeptide. These polypeptides canalso be obtained in any of a number of procedures well known in the art.One method of producing a polypeptide is to link two peptides orpolypeptides together by protein chemistry techniques. For example,peptides or polypeptides can be chemically synthesized using currentlyavailable laboratory equipment using either Fmoc(9-fluorenylmethyloxycarbonyl) or Boc (tert-butyloxycarbonoyl)chemistry. (Applied Biosystems, Inc., Foster City, Calif.). One skilledin the art can readily appreciate that a peptide or polypeptidecorresponding to a particular protein can be synthesized by standardchemical reactions. For example, a peptide or polypeptide can besynthesized and not cleaved from its synthesis resin whereas the otherfragment of a hybrid peptide can be synthesized and subsequently cleavedfrom the resin, thereby exposing a terminal group which is functionallyblocked on the other fragment. By peptide condensation reactions, thesetwo fragments can be covalently joined via a peptide bond at theircarboxyl and amino termini, respectively, to form a larger polypeptide.(Grant, ASynthetic Peptides: A User Guide, “W.H. Freeman and Co., N.Y.(1992) and Bodansky and Trost, Ed., APrinciples of Peptide Synthesis,”Springer-Verlag Inc., N.Y. (1993)). Alternatively, the peptide orpolypeptide can be independently synthesized in vivo as described above.Once isolated, these independent peptides or polypeptides may be linkedto form a larger protein via similar peptide condensation reactions.

For example, enzymatic ligation of cloned or synthetic peptide segmentscan allow relatively short peptide fragments to be joined to producelarger peptide fragments, polypeptides or whole protein domains(Abrahmsen et al. Biochemistry, 30:4151 (1991)). Alternatively, nativechemical ligation of synthetic peptides can be utilized to syntheticallyconstruct large peptides or polypeptides from shorter peptide fragments.This method consists of a two-step chemical reaction (Dawson et al.“Synthesis of Proteins by Native Chemical Ligation,” Science,266:776-779 (1994)). The first step is the chemoselective reaction of anunprotected synthetic peptide--thioester with another unprotectedpeptide segment containing an amino-terminal Cys residue to give athioester-linked intermediate as the initial covalent product. Without achange in the reaction conditions, this intermediate undergoesspontaneous, rapid intramolecular reaction to form a native peptide bondat the ligation site. Application of this native chemical ligationmethod to the total synthesis of a protein molecule is illustrated bythe preparation of human interleukin 8 (IL-8) (Clark-Lewis et al. FEBSLett., 307:97 (1987), Clark-Lewis et al., J. Biol. Chem., 269:16075(1994), Clark-Lewis et al. Biochemistry, 30:3128 (1991), and Rajarathnamet al. Biochemistry, 29:1689 (1994)).

Alternatively, unprotected peptide segments can be chemically linkedwhere the bond formed between the peptide segments as a result of thechemical ligation is an unnatural (non-peptide) bond (Schnolzer et al.Science, 256:221 (1992)). This technique has been used to synthesizeanalogs of protein domains as well as large amounts of relatively pureproteins with full biological activity (deLisle Milton et al.ATechniques in Protein Chemistry IV, Academic Press, New York, pp.257-267 (1992)).

The present invention also provides a composition comprising a complexbetween IL-22R1 and IL-20R2. Therefore, compositions of the presentinvention include complexes formed by the interaction of IL-22R1 withIL-20R2. Further provided is a composition comprising a complex betweenIL-20R1 and IL-20R2. Therefore, compositions of the present inventioninclude complexes formed by the interaction of IL-20R1 with IL-20R2.Also provided by this invention is a composition comprising a complexbetween Mob-5 (IL-24) and IL-22R1 and IL-20R2 as well as a compositioncomprising a complex between Mob-5 (IL-24) and IL-20R1 and IL-20R2.Further provided is a composition comprising a complex between Mob-5(IL-24) and IL-22R1. Also provided is a composition comprising a complexbetween Mob-5 (IL-24) and IL-20R2. The invention also provides acomposition comprising a complex between Mob-5(IL-24) and IL-20R1.

The present invention also provides a composition comprising a solubleform of the Mob-5 receptor formed by the extracellular portions ofIL-22R1 and IL-20R2 or the extracellular portions of IL-20R1 andIL-20R2. For example, the present invention provides amino acids 1-226of IL-22R1 (SEQ ID NO: 1) as a soluble extracellular portion of IL-22R1that can be combined with amino acids 1-229 of IL-20R2 (SEQ ID NO: 2) toform a soluble Mob-5 receptor. This invention further provides aminoacids 1-248 of IL-20R1 (SEQ ID NO: 3) that can be combined with aminoacids 1-229 of IL-20R2 (SEQ ID NO: 2) to form a soluble Mob-5 receptor.Fragments of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 are alsoprovided which can be combined to form a soluble Mob-5 receptor. Thepresent invention also provides the use of the individual receptorsubunits, i.e. SEQ ID NO: 1, SEQ ID NO:2 or SEQ ID NO: 3 and fragmentsthereof as soluble receptors for Mob-5. One of skill in the art wouldknow how to construct soluble Mob-5 receptors and test these solubleMob-5 receptors for Mob-5 binding as described herein and as known inthe art in order to obtain soluble forms of the Mob-5 receptor thatretain Mob-5 binding ability. These soluble forms of the Mob-5 receptorcan be utilized to block Mob-5 binding to Mob-5 receptors by competingwith Mob-5 receptors for the binding of Mob-5.

Mob-5 Receptor Nucleic Acids

The present invention provides an isolated nucleic acid encoding thereceptors, receptor subunits and peptides of the invention. For example,the invention provides an isolated nucleic acid encoding IL-22R1 andIL-20R2. The present invention also provides an isolated nucleic acidencoding IL-20R1 and L-20R2. Furthermore, the present invention providesnucleic acids encoding IL-22R1, IL-20R1 or IL-20R2 and fragmentsthereof.

A nucleic acid of this invention encodes both proteins (IL-22R1 andIL-20R2) that comprise a heterodimeric receptor for Mob-5 (IL-24).Another nucleic acid of this invention encodes both proteins (IL-20R1and IL-20R2) that comprise another heterodimeric receptor for Mob-5(IL-24). As used herein, the term Anucleic acid@ refers to single-ormultiple stranded molecules which may be DNA or RNA, or any combinationthereof, including modifications to those nucleic acids. The nucleicacid may represent a coding strand or its complement, or any combinationthereof. Nucleic acids may be identical in sequence to the sequenceswhich are naturally occurring for any of the genes discussed herein ormay include alternative codons which encode the same amino acid as thatwhich is found in the naturally occurring sequence. These nucleic acidscan also be modified from their typical structure. Such modificationsinclude, but are not limited to, methylated nucleic acids, thesubstitution of a non-bridging oxygen on the phosphate residue witheither a sulfur (yielding phosphorothioate deoxynucleotides), selenium(yielding phosphorselenoate deoxynucleotides), or methyl groups(yielding methylphosphonate deoxynucleotides).

The nucleic acids provided for by the present invention may be obtainedin any number of ways. For example, DNA molecules encoding IL-22R1,IL-22R1 or IL-20R2 can be obtained from commercial sources as well asfrom academic laboratories. Also, a DNA molecule encoding IL-22R1 orIL-20R2 protein can be isolated from the organism in which it isnormally found. For example, a genomic DNA or cDNA library can beconstructed and screened for the presence of the gene or nucleic acid ofinterest. Methods of constructing and screening such libraries are wellknown in the art and kits for performing the construction and screeningsteps are commercially available (for example, Stratagene CloningSystems, La Jolla, Calif.). Once isolated, the gene or nucleic acid canbe directly cloned into an appropriate vector, or if necessary, bemodified to facilitate the subsequent cloning steps. Such modificationsteps are routine, an example of which is the addition ofoligonucleotide linkers which contain restriction sites to the terminiof the nucleic acid. General methods are set forth in Sambrook et al.,AMolecular Cloning, a Laboratory Manual,@ Cold Spring Harbor LaboratoryPress (1989).

Once the gene or nucleic acid sequence of the desired protein isobtained, the sequence encoding specific amino acids can be modified orchanged at any particular amino acid position by techniques well knownin the art. For example, PCR primers can be designed which span theamino acid position or positions and which can substitute any amino acidfor another amino acid. Then a nucleic acid can be amplified andinserted into the wild-type protein coding sequence in order to obtainany of a number of possible combinations of amino acids at any positionof the protein. Alternatively, one skilled in the art can introducespecific mutations at any point in a particular nucleic acid sequencethrough techniques for point mutagenesis. General methods are set forthin Smith, M “In vitro mutagenesis” Ann. Rev. Gen., 19:423-462 (1985) andZoller, M. J. “New molecular biology methods for protein engineering”Curr. Opin. Struct. Biol., 1:605-610 (1991). Techniques such as thesecan also be used to modify the genes or nucleic acids in regions otherthan the coding regions, such as the promoter regions for IL-22R1 andIL-20R2. Likewise, these techniques can be used to alter the codingsequence without altering the amino acid sequence that is encoded.

Another example of a method of obtaining a DNA molecule encoding aspecific Mob-5 receptor protein is to synthesize a recombinant DNAmolecule which encodes the Mob-5 receptor proteins. For example,oligonucleotide synthesis procedures are routine in the art andoligonucleotides coding for a particular protein region are readilyobtainable through automated DNA synthesis. A nucleic acid for onestrand of a double-stranded molecule can be synthesized and hybridizedto its complementary strand. One can design these oligonucleotides suchthat the resulting double-stranded molecule has either internalrestriction sites or appropriate 5′ or 3′ overhangs at the termini forcloning into an appropriate vector. Double-stranded molecules coding forrelatively large proteins can readily be synthesized by firstconstructing several different double-stranded molecules that code forparticular regions of the protein, followed by ligating these DNAmolecules together. For example, Cunningham, et al., AReceptor andAntibody Epitopes in Human Growth Hormone Identified by Homolog-ScanningMutagenesis,@ Science, 243:1330-1336 (1989), have constructed asynthetic gene encoding the human growth hormone gene by firstconstructing overlapping and complementary synthetic oligonucleotidesand ligating these fragments together. See also, Ferretti, et al., Proc.Nat. Acad. Sci. 82:599-603 (1986), wherein synthesis of a 1057 base pairsynthetic bovine rhodopsin gene from synthetic oligonucleotides isdisclosed. By constructing a Mob-5 receptor protein, i.e. IL-22R1 and/orIL-20R2, in this manner, one skilled in the art can readily obtain anyparticular Mob-5 receptor protein with desired amino acids at anyparticular position or positions within the Mob-5 receptor protein. Seealso, U.S. Pat. No. 5,503,995 which describes an enzyme templatereaction method of making synthetic genes. Techniques such as this areroutine in the art and are well documented. These nucleic acids orfragments of a nucleic acid encoding Mob-5 receptor proteins can then beexpressed in vivo or in vitro as discussed below.

The invention also provides for the isolated nucleic acids encodingIL-22R1 and IL-20R2 in a vector suitable for expressing the nucleicacid. The invention also contemplates a vector comprising an isolatednucleic acid encoding IL-22R1 and a vector comprising an isolatednucleic acid encoding IL-20R2 for contransfection of cells to obtainrecombinant cells expressing both IL-22R1 and IL-20R2.

The invention also provides for the isolated nucleic acids encodingIL-20R1 and IL-20R2 in a vector suitable for expressing the nucleicacid. Also provided by this invention is a vector comprising an isolatednucleic acid encoding IL-20R1 and a vector comprising an isolatednucleic acid encoding IL-20R2 for contransfection of cells to obtainrecombinant cells expressing both IL-20R1 and IL-20R2.

Once a nucleic acid encoding IL-22R1 and IL-20R2, or a nucleic acidencoding IL-20R1 and IL-20R2, or a region of that nucleic acid, isconstructed, modified, or isolated, that nucleic acid can then be clonedinto an appropriate vector, which can direct the in vivo or in vitrosynthesis of that wild-type and/or modified Mob-5 receptor proteins. Thevector is contemplated to have the necessary functional elements thatdirect and regulate transcription of the inserted gene, or nucleic acid.These functional elements include, but are not limited to, a promoter,regions upstream or downstream of the promoter, such as enhancers thatmay regulate the transcriptional activity of the promoter, an origin ofreplication, appropriate restriction sites to facilitate cloning ofinserts adjacent to the promoter, antibiotic resistance genes or othermarkers which can serve to select for cells containing the vector or thevector containing the insert, RNA splice junctions, a transcriptiontermination region, or any other region which may serve to facilitatethe expression of the inserted gene or hybrid gene. (See generally,Sambrook et al.).

There are numerous E. coli (Escherichia coli) expression vectors knownto one of ordinary skill in the art which are useful for the expressionof the nucleic acid insert. Other microbial hosts suitable for useinclude bacilli, such as Bacillus subtilis, and otherenterobacteriaceae, such as Salmonella, Serratia, and variousPseudomonas species. In these prokaryotic hosts one can also makeexpression vectors, which will typically contain expression controlsequences compatible with the host cell (e.g., an origin ofreplication). In addition, any number of a variety of well-knownpromoters will be present, such as the lactose promoter system, atryptophan (Trp) promoter system, a beta-lactamase promoter system, or apromoter system from phage lambda. The promoters will typically controlexpression, optionally with an operator sequence, and have ribosomebinding site sequences for example, for initiating and completingtranscription and translation. If necessary, an amino terminalmethionine can be provided by insertion of a Met codon 5′ and in-framewith the downstream nucleic acid insert. Also, the carboxy-terminalextension of the nucleic acid insert can be removed using standardoligonucleotide mutagenesis procedures.

Additionally, yeast expression can be used. There are several advantagesto yeast expression systems. First, evidence exists that proteinsproduced in a yeast secretion systems exhibit correct disulfide pairing.Second, post-translational glycosylation is efficiently carried out byyeast secretory systems. The Saccharomyces cerevisiaepre-pro-alpha-factor leader region (encoded by the MF″-1 gene) isroutinely used to direct protein secretion from yeast. (Brake, et al., A%-Factor-Directed Synthesis and Secretion of Mature Foreign Proteins inSaccharomyces cerevisiae.@ Proc. Nat. Acad. Sci., 81:4642-4646 (1984)).The leader region of pre-pro-alpha-factor contains a signal peptide anda pro-segment which includes a recognition sequence for a yeast proteaseencoded by the KEX2 gene: this enzyme cleaves the precursor protein onthe carboxyl side of a Lys-Arg dipeptide cleavage signal sequence. Thenucleic acid coding sequence can be fused in-frame to thepre-pro-alpha-factor leader region. This construct is then put under thecontrol of a strong transcription promoter, such as the alcoholdehydrogenase I promoter or a glycolytic promoter. The nucleic acidcoding sequence is followed by a translation termination codon which isfollowed by transcription termination signals. Alternatively, thenucleic acid coding sequences can be fused to a second protein codingsequence, such as Sj26 or beta-galactosidase, used to facilitatepurification of the fusion protein by affinity chromatography. Theinsertion of protease cleavage sites to separate the components of thefusion protein is applicable to constructs used for expression in yeast.Efficient post translational glycosylation and expression of recombinantproteins can also be achieved in Baculovirus systems.

Mammalian cells permit the expression of proteins in an environment thatfavors important post-translational modifications such as folding andcysteine pairing, addition of complex carbohydrate structures, andsecretion of active protein. Vectors useful for the expression of activeproteins in mammalian cells are characterized by insertion of theprotein coding sequence between a strong viral promoter and apolyadenylation signal. The vectors can contain genes conferringhygromycin resistance, gentamicin resistance, or other genes orphenotypes suitable for use as selectable markers, or methotrexateresistance for gene amplification. The chimeric protein coding sequencecan be introduced into a Chinese hamster ovary (CHO) cell line using amethotrexate resistance-encoding vector, or other cell lines usingsuitable selection markers. Presence of the vector DNA in transformedcells can be confirmed by Southern blot analysis. Production of RNAcorresponding to the insert coding sequence can be confirmed by Northernblot analysis. A number of other suitable host cell lines capable ofsecreting intact human proteins have been developed in the art, andinclude the CHO cell lines, HeLa cells, myeloma cell lines, Jurkatcells, etc. Expression vectors for these cells can include expressioncontrol sequences, such as an origin of replication, a promoter, anenhancer, and necessary information processing sites, such as ribosomebinding sites, RNA splice sites, polyadenylation sites, andtranscriptional terminator sequences. Preferred expression controlsequences are promoters derived from immunoglobulin genes, SV40,Adenovirus, Bovine Papilloma Virus, etc. The vectors containing thenucleic acid segments of interest can be transferred into the host cellby well-known methods, which vary depending on the type of cellularhost. For example, calcium chloride transformation is commonly utilizedfor prokaryotic cells, whereas calcium phosphate, DEAE dextran, orlipofectin mediated transfection or electroporation may be used forother cellular hosts.

Alternative vectors for the expression of genes or nucleic acids inmammalian cells, those similar to those developed for the expression ofhuman gamma-interferon, tissue plasminogen activator, clotting FactorVIII, hepatitis B virus surface antigen, protease Nexin 1, andeosinophil major basic protein, can be employed. Further, the vector caninclude CMV promoter sequences and a polyadenylation signal availablefor expression of inserted nucleic acids in mammalian cells (such asCOS-7).

Insect cells also permit the expression of mammalian proteins.Recombinant proteins produced in insect cells with baculovirus vectorsundergo post-translational modifications similar to that of wild-typeproteins. Briefly, baculovirus vectors useful for the expression ofactive proteins in insect cells are characterized by insertion of theprotein coding sequence downstream of the Autographica californicanuclear polyhedrosis virus (AcNPV) promoter for the gene encodingpolyhedrin, the major occlusion protein. Cultured insect cells such asSpodoptera frugiperda cell lines are transfected with a mixture of viraland plasmid DNAs and the viral progeny are plated. Deletion orinsertional inactivation of the polyhedrin gene results in theproduction of occlusion negative viruses which form plaques that aredistinctively different from those of wild-type occlusion positiveviruses. These distinctive plaque morphologies allow visual screeningfor recombinant viruses in which the AcNPV gene has been replaced with ahybrid gene of choice.

The invention also provides for the vectors containing the contemplatednucleic acids in a host suitable for expressing the nucleic acids. Thevectors containing the nucleic acid segments of interest can betransferred into host cells by well-known methods, which vary dependingon the type of cellular host. For example, calcium chloridetransformation, transduction, and electroporation are commonly utilizedfor prokaryotic cells, whereas calcium phosphate, DEAE dextran, orlipofection mediated transfection or electroporation may be used forother cellular hosts.

Alternatively, the genes or nucleic acids of the present invention canbe operatively linked to one or more of the functional elements thatdirect and regulate transcription of the inserted gene as discussedabove and the gene or nucleic acid can be expressed. For example, a geneor nucleic acid can be operatively linked to a bacterial or phagepromoter and used to direct the transcription of the gene or nucleicacid in vitro. A further example includes using a gene or nucleic acidprovided herein in a coupled transcription-translation system where thegene directs transcription and the RNA thereby produced is used as atemplate for translation to produce a polypeptide. One skilled in theart will appreciate that the products of these reactions can be used inmany applications such as using labeled RNAs as probes and usingpolypeptides to generate antibodies or in a procedure where thepolypeptides are being administered to a cell or a patient.

Expression of the gene or nucleic acid, in combination with a vector,can be by either in vivo or in vitro. In vivo synthesis comprisestransforming prokaryotic or eukaryotic cells that can serve as hostcells for the vector. Alternatively, expression of the gene or nucleicacid can occur in an in vitro expression system. For example, in vitrotranscription systems are commercially available which are routinelyused to synthesize relatively large amounts of mRNA. In such in vitrotranscription systems, the nucleic acid encoding the Mob-5 receptorproteins would be cloned into an expression vector adjacent to atranscription promoter. For example, the Bluescript II cloning andexpression vectors contain multiple cloning sites which are flanked bystrong prokaryotic transcription promoters. (Stratagene Cloning Systems,La Jolla, Calif.). Kits are available which contain all the necessaryreagents for in vitro synthesis of an RNA from a DNA template such asthe Bluescript vectors. (Stratagene Cloning Systems, La Jolla, Calif.).RNA produced in vitro by a system such as this can then be translated invitro to produce the desired Mob-5 receptor protein. (Stratagene CloningSystems, La Jolla, Calif.). High quantity expression and production ofthe Mob-5 receptor protein can also be achieved by transgenic animaltechnology by which animals can be made to produce Mob-5 receptorprotein, or soluble Mob-5 receptor in serum, milk, etc in large amounts.

Mob-5 Receptor Antibodies

Also provided herein are purified antibodies that selectively orspecifically bind to the complexes provided and contemplated herein.Antibodies that specifically bind to the complexes do not bind toindividual components of the complex. For example, purified antibodieswhich selectively or specifically bind to a complex between IL-22R1 andIL-20R2 do not bind to IL-22R1 in the absence of Il-20R2 nor does itbind to IL-20R2 in the absence of Il-22R1. Therefore, an antibody thatbinds to the complex formed between IL-22R1 and IL-20R2 is specific forthe Mob-5 receptor that is formed by the dimerization of these twoproteins.

Antibodies that bind to a complex between Mob-5 (IL-24) and IL-22R1 andIL-20R2 are also provided. Also provided are antibodies that bind to acomplex between Mob-5 (IL-24) and IL-20R1 and IL-20R2. Further providedis an antibody that binds to a complex between Mob-5 (IL-24) andIL-22R1. Also provided is an antibody that binds to a complex betweenMob-5 (IL-24) and IL-20R2. The invention also provides an antibody thatbinds to a complex between Mob-5 (IL-24) and IL-20R1. Antibodies thatbind to Mob-5, IL-20R1, IL-22R1 or IL-20R2 or fragments thereof are alsocontemplated herein.

Antibodies can be made by many well-known methods (See, e.g. Harlow andLane, “Antibodies; A Laboratory Manual” Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y., (1988)). Briefly, purified antigen can beinjected into an animal in an amount and in intervals sufficient toelicit an immune response. Antibodies can either be purified directly,or spleen cells can be obtained from the animal. The cells can thenfused with an immortal cell line and screened for antibody secretion.The antibodies can be used to screen nucleic acid clone libraries forcells secreting the antigen. Those positive clones can then besequenced. (See, for example, Kelly et al. Bio/Technology, 10:163-167(1992); Bebbington et al. Bio/Technology, 10:169-175 (1992)). Humanizedand chimeric antibodies are also contemplated in this invention.Heterologous antibodies can be made by well known methods (See, forexample, U.S. Pat. Nos. 5,545,806, 5,569,825, 5,625,126, 5,633,425,5,661,016, 5,770,429, 5,789,650, and 5,814,318).

The phrase “specifically binds” with the polypeptide refers to a bindingreaction which is determinative of the presence of the protein in aheterogeneous population of proteins and other biologics. Thus, underdesignated immunoassay conditions, the specified antibodies bound to aparticular protein do not bind in a significant amount to other proteinspresent in the sample. Selective binding to an antibody under suchconditions may require an antibody that is selected for its specificityfor a particular protein. A variety of immunoassay formats may be usedto select antibodies that selectively bind with a particular protein.For example, solid-phase ELISA immunoassays are routinely used to selectantibodies selectively immunoreactive with a protein. See Harlow andLane “Antibodies, A Laboratory Manual” Cold Spring Harbor Publications,New York, (1988), for a description of immunoassay formats andconditions that could be used to determine selective binding.

The term “antibody” is used herein in a broad sense and includes intactimmunoglobulin molecules and fragments or polymers of thoseimmunoglobulin molecules, so long as they exhibit any of the desiredproperties described herein. Antibodies are typically proteins whichexhibit binding specificity to a specific antigen. Native antibodies areusually heterotetrameric glycoproteins, composed of two light (L) chainsand two heavy (H) chains. The heavy and light chains are typicallyidentical, but not necessarily so. Typically, each light chain is linkedto a heavy chain by one or more covalent disulfide bond, while thenumber of disulfide linkages varies between the heavy chains ofdifferent immunoglobulin isotypes. Each heavy and light chain alsotypically has regularly spaced intrachain disulfide bridges. Each heavychain typically has at one end a variable domain (V(H)) followed by anumber of constant domains. Each light chain typically has a variabledomain at one end (V(L)) and a constant domain at its other end; theconstant domain of the light chain is typically aligned with the firstconstant domain of the heavy chain, and the light chain variable domainis typically aligned with the variable domain of the heavy chain.Particular amino acid residues are believed to form an interface betweenthe light and heavy chain variable domains. The light chains ofantibodies from any vertebrate species can be assigned to one of twoclearly distinct types, called kappa (κ) and lambda (λ), based on theamino acid sequences of their constant domains. Depending on the aminoacid sequence of the constant domain of their heavy chains,immunoglobulins can typically be assigned to different classes. Thereare approximately five major classes of immunoglobulins: IgA, IgD, IgE,IgG and IgM, and several of these may be further divided into subclasses(isotypes), e.g., IgG-1, IgG-2, IgG-3, and IgG-4; IgA-1 and IgA-2. Theheavy chain constant domains that correspond to the different classes ofimmunoglobulins are called α, δ, ε, γ, and μ, respectively.

The term “variable” is used herein to describe certain portions of thevariable domains which differ in sequence among antibodies and are usedin the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not usually evenlydistributed through the variable domains of antibodies. It is typicallyconcentrated in three segments called complementarity determiningregions (CDRs) or hypervariable regions both in the light chain and theheavy chain variable domains. The more highly conserved portions of thevariable domains are called the framework (FR). The variable domains ofnative heavy and light chains each comprise four FR regions, largelyadopting a β-sheet configuration, connected by three CDRs, which formloops connecting, and in some cases forming part of, the β.-sheetstructure. The CDRs in each chain are held together in close proximityby the FR regions and, with the CDRs from the other chain, contribute tothe formation of the antigen binding site of antibodies (see Kabat E. A.et al., “Sequences of Proteins of Immunological Interest” NationalInstitutes of Health, Bethesda, Md. (1987)). The constant domains arenot involved directly in binding an antibody to an antigen, but exhibitvarious effector functions, such as participation of the antibody inantibody-dependent cellular toxicity.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a substantially homogeneous population of antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. The monoclonal antibodies herein specifically include“chimeric” antibodies in which a portion of the heavy and/or light chainmay be identical with or homologous to corresponding sequences inantibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) may be identical with or homologous to corresponding sequencesin antibodies derived from another species or belonging to anotherantibody class or subclass, as well as fragments of such antibodies, solong as they exhibit the desired antagonistic activity (See, U.S. Pat.No. 4,816,567 and Morrison et al., Proc. Natl. Acad. Sci. USA,81:6851-6855 (1984)).

The antibody (either polyclonal or monoclonal) can be raised to any ofthe polypeptides provided and contemplated herein, both naturallyoccurring and recombinant polypeptides, and immunogenic fragments,thereof. The antibody can be used in techniques or procedures such asdiagnostics, treatment, or vaccination. Anti-idiotypic antibodies andaffinity matured antibodies are also considered.

The antibodies of the invention include anti-Mob 5 receptor antibodiesand anti-Mob 5 antibodies that may further comprise humanized antibodiesor human antibodies. Humanized forms of non-human (e.g., murine)antibodies are chimeric immunoglobulins, immunoglobulin chains orfragments thereof (such as Fv, Fab, Fab′ or other antigen-bindingsubsequences of antibodies) which contain minimal sequence derived fromnon-human immunoglobulin. Humanized antibodies include humanimmunoglobulins (recipient antibody) in which residues from acomplementarity determining region (CDR) of the recipient are replacedby residues from a CDR of a non-human species (donor antibody) such asmouse, rat or rabbit having the desired specificity, affinity andcapacity. In some instances, Fv framework residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin (Jones et al., Nature, 321:522-525 (1986), Reichmann etal., Nature, 332:323-327 (1988), and Presta, Curr. Op. Struct. Biol.,2:593-596 (1992)).

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers(Jones et al., Nature, 321:522-525 (1986), Riechmann et al., Nature,332:323-327 (1988), Verhoeyen et al., Science, 239:1534-1536 (1988)), bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important in order to reduceantigenicity. According to the “best-fit” method, the sequence of thevariable domain of a rodent antibody is screened against the entirelibrary of known human variable domain sequences. The human sequencewhich is closest to that of the rodent is then accepted as the humanframework (FR) for the humanized antibody (Sims et al., J. Immunol.,151:2296 (1993) and Chothia et al, J. Mol. Biol., 196:901 (1987)).Another method uses a particular framework derived from the consensussequence of all human antibodies of a particular subgroup of light orheavy chains. The same framework may be used for several differenthumanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285(1992); Presta et al., J. Immunol., 151:2623 (1993)).

It is further important that antibodies be humanized with retention ofhigh affinity for the antigen and other favorable biological properties.To achieve this goal, according to a preferred method, humanizedantibodies are prepared by a process of analysis of the parentalsequences and various conceptual humanized products using threedimensional models of the parental and humanized sequences. Threedimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese, displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the consensus and import sequence so that thedesired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the CDR residues aredirectly and most substantially involved in influencing antigen binding(see, WO 94/04679 published 3 Mar. 1994).

Transgenic animals (e.g., mice) that are capable, upon immunization, ofproducing a full repertoire of human antibodies in the absence ofendogenous immunoglobulin production can be employed. For example, ithas been described that the homozygous deletion of the antibody heavychain joining region (J(H)) gene in chimeric and germ-line mutant miceresults in complete inhibition of endogenous antibody production.Transfer of the human germ-line immunoglobulin gene array in suchgerm-line mutant mice will result in the production of human antibodiesupon antigen challenge (see, e.g., Jakobovits et al., Proc. Natl. Acad.Sci. USA, 90:2551-255 (1993); Jakobovits et al., Nature, 362:255-258(1993); Bruggermann et al., Year in Immuno., 7:33 (1993)). Humanantibodies can also be produced in phage display libraries [Hoogenboomet al., J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol.,222:581 (1991)]. The techniques of Cote et al. and Boerner et al. arealso available for the preparation of human monoclonal antibodies (Coleet al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77(1985) and Boerner et al., J. Immunol., 147(1):86-95 (1991)].

The purified antibodies of this invention include monoclonal antibodieswhich can be used for diagnostic or analytical purposes. For example,the monoclonal antibody could be utilized in a clinical testing kit tomonitor levels of Mob-5 receptor in human tissues or secretions.

Detection Methods

The present invention also provides a method of detecting the Mob-5receptor comprising, contacting a cell with a labeled Mob-5, detectingthe binding of the labeled Mob-5 to the cell, wherein binding of thelabeled Mob-5 to the cell indicates the cell is a Mob-5 receptorexpressing cell, thus detecting the Mob-5 receptor.

In the methods of the present invention, the labeled Mob-5 includes¹²⁵I-labeled Mob-5, a Mob-5-alkaline phosphatase fusion protein(Mob-5-AP), a Mob-5-IGg Fc fusion protein, a horse radish peroxidase(HRP) labeled Mob-5, a biotin labeled Mob-5 and any other Mob-5 linkedto a detectable moiety. One of skill in the art would know how tomeasure the amounts of labeled Mob-5 in the methods of the presentinvention based on the teachings set forth in the Examples and thosewell known in the art.

The present invention further provides a method of detecting Mob-5activation of a Mob-5 receptor comprising: a) contacting a cell withMob-5; b) measuring STAT activation, wherein an increase in STATactivation in the cell of step a) as compared to STAT activation in acell not contacted with Mob-5 indicates Mob-5 activation of a Mob-5receptor. One of skill in the art would know how to measure STATactivation based on the teachings set forth in the Examples as well asteachings known in the art.

In the methods of the present invention, the Mob-5 receptor can be theIL-20R1/IL-20R2 heterodimer or the IL-22R1/IL-20R2 heterodimer or both.

Methods of Inhibiting Mob-5 Receptors

Also provided by the present invention is a method for inhibiting amob-S induced cellular transformation phenotype comprising inhibitingthe Mob-5 receptor in a cell so as to inhibit the expression of themob-S induced cellular transformation phenotype.

Further provided by the present invention is a method for inhibiting aMob-5 induced cancer comprising inhibiting the Mob-5 receptor in a cellso as to inhibit the Mob-5 induced cancer.

In this invention, Acellular transformation phenotype@ means that cellshave undergone at least some transformation characterized by one or moremorphologic or biochemical changes such as loss of contact inhibition,increased rate of glycolysis, alterations of the cell surface and otherchanges that would be known to one skilled in the art. Other changesinclude increased ability to grow in soft agar and increasedtumorogenicity in nude mice.

In this invention, Ainhibition of cancer@ or Ainhibition of cancerformation@ means partial or total killing of cancerous cells, reductionin tumor size, disappearance of a tumor, inhibition of tumor growth,inhibition of vascularization, inhibition of cellular proliferation, aninduction in dormancy or an apparent induction of dormancy, or adecreased metastasis of a tumor or a tumor cell. These mechanisms ofaction are only exemplary of the ways that an inhibitor of the Mob-5receptor can inhibit or treat cancer. The methods of the presentinvention can be utilized to inhibit or treat various types of cancersincluding, but not limited to, pancreatic cancer, skin cancer, larynxcancer, lung cancer, breast cancer, adrenocarcinoma and colon cancer.

The term Ainhibiting@ is familiar to one skilled in the art and is usedherein to describe any compound or composition which inhibits ordecreases the expression or activity of a Mob-5 receptor. The degree ofinhibition does not have to be complete, such as completely inhibitingthe expression or activity of a Mob-5 receptor and therefore comprisesany inhibition of the expression or activity of the Mob-5 receptorrelative to the expression of the Mob-5 receptor in a similarenvironment in the absence of the inhibiting compound. Inhibition canoccur in many ways such by inhibiting gene expression, inhibiting Mob-5binding to Mob-5 receptor, inhibiting Mob-5 receptor activation,administering an antibody to a Mob-5 receptor, administering an antibodyto a Mob-5 receptor subunit and by other methods known in the art.

The cellular transformation phenotype and/or cancer of this inventioncan be inhibited by disrupting dimerization between IL-22R1 and IL-20R2in order to prevent a Mob-5 receptor from forming, thus preventingbinding of Mob-5 to its receptor and effecting inhibition of a cellulartransformation phenotype. For example, an anti-IL-22R1 antibody or ananti-IL-20R2 antibody can be utilized to disrupt dimerization. Fragmentsof IL-22R1 and IL-20R2 can also be utilized to disrupt dimerization. Oneof skill in the art could identify the contact points between thesubunits and design peptides that would disrupt dimerization. Forexample, crystal structures of IL-22R1 and Il-20R2 and their complexesmay be utilized to design molecules that disrupt Mob-5 binding to itsreceptor.

The cellular transformation phenotype and/or cancer of this inventioncan also be inhibited by disrupting dimerization between IL-20R1 andIL-20R2 in order to prevent a Mob-5 receptor from forming, thuspreventing binding of Mob-5 to its receptor and effecting inhibition ofa cellular transformation phenotype. For example, an anti-IL-20R1antibody or an anti-IL-20R2 antibody can be utilized to disruptdimerization. Fragments of IL-20R1 and IL-20R2 can also be utilized todisrupt dimerization. One of skill in the art could identify the contactpoints between the subunits and design peptides that would disruptdimerization. For example, crystal structures of IL-20R1 and Il-20R2 andtheir complexes may be utilized to design molecules that disrupt Mob-5binding to its receptor.

Alternatively, an inhibitor of the Mob-5 receptor can be administered toa cell. An “inhibitor” is defined as a compound that binds a Mob-5receptor including antibodies, that prevents an activity of a Mob-5receptor. Upon binding to the receptor, the inhibitor can disrupt orprevent Mob-5 binding to the Mob-5 receptor. The inhibitor can be anantibody, either polyclonal or monoclonal, that specifically binds to aMob-5 receptor, a ligand that binds to a Mob-5 receptor, a polypeptidethat binds to a Mob-5 receptor or a compound that binds to a Mob-5receptor. The inhibitor can also be an antibody, either eitherpolyclonal or monoclonal, that specifically binds to a Mob-5 (ligand forthe Mob-5 receptor), a ligand that binds to a Mob-5, a polypeptide thatbinds to Mob-5 or a compound that binds to a Mob-5 and thus prevents theinteraction of Mob-5 with the Mob-5 receptor. Anti-idiotypic antibodies,affinity matured antibodies and humanized antibodies of Mob-5 receptorand Mob-5 are also considered. Other inhibitors include, but are notlimited to molecules or compounds designed to block the binding of Mob-5to the Mob-5 receptor. The inhibitor can be a whole protein or afragment of a protein that inhibits binding, thus preventing Mob-5receptor activation. The inhibitors can also be soluble fragments of theMob-5 receptor that bind to Mob-5 and blocks the binding of Mob-5 tocell surface Mob-5 receptors.

A Mob-5 receptor can also be inhibited by administering to the cell analtered Mob-5 protein which binds to the Mob-5 receptor, whereby bindingof the altered Mob-5 to the receptor inhibits the binding of Mob-5 tothe receptor, thereby inhibiting the expression of a mob-5 inducedcellular transformation phenotype.

The cellular transformation phenotype and/or cancer of this inventioncan be inhibited by a nucleic acid antisense to the Mob-5 receptor. Forexample, a nucleic acid antisense to the nucleic acid encoding IL-22R1and/or a nucleic acid antisense to the nucleic acid encoding IL-20R2 canbe utilized to inhibit expression of the genes that comprise a Mob-5receptor. A nucleic acid antisense to the nucleic acid encoding IL-20R1and/or a nucleic acid antisense to the nucleic acid encoding IL-20R2 canalso be utilized to inhibit expression of the genes that comprise aMob-5 receptor. Antisense technology is well known in the art anddescribes a mechanism whereby a nucleic acid comprising a nucleotidesequence which is in a complementary, Aantisense@ orientation withrespect to a coding or Asense@ sequence of an endogenous gene, isintroduced into a cell, whereby a duplex may form between the antisensesequence and its complementary sense sequence. The formation of thisduplex may result in inactivation of the endogenous gene.

For example, the antisense nucleic acid can inhibit gene expression byforming an RNA/RNA duplex between the antisense RNA and the RNAtranscribed from a target gene. The precise mechanism by which thisduplex formation decreases the production of the protein encoded by theendogenous gene most likely involves binding of complementary regions ofthe normal sense mRNA and the antisense RNA strand with duplex formationin a manner that blocks RNA processing and translation. Alternativemechanisms include the formation of a triplex between the antisense RNAand duplex DNA or the formation of a DNA-RNA duplex with subsequentdegradation of DNA-RNA hybrids by RNAse H. Furthermore, an antisenseeffect can result from certain DNA-based oligonucleotides viatriple-helix formation between the oligomer and double-stranded DNAwhich results in the repression of gene transcription. Antisense nucleicacid can be produced for any relevant endogenous gene for which thecoding sequence has been or can be determined according to well knownmethods.

A nucleic acid encoding an antisense RNA can be selected based on theprotein desired to be inhibited or decreased in cells, by providing anRNA that will selectively bind to the cellular mRNA encoding suchprotein. Binding of the antisense molecule to the target mRNA mayincapacitate the mRNAs, thus preventing its translation into afunctional protein. The antisense RNA/mRNA complexes can then become atarget for RNAse-H and are eventually degraded by the host cell RNAse-H.Control regions, such as enhancers and promoters, can be selected forantisense RNA targeting according to the cell or tissue in which it isto be expressed, as is known in the art. Preferable antisense-encodingconstructs can encode full-length complements to target sequences;however, smaller length sequences down to oligonucleotide size can beutilized. For example, the antisense-encoding constructs can encodefull-length complements to the mob-5 gene, smaller length sequences oroligonucleotide sequences.

The present invention further provides a method of inhibiting a cellulartransformation phenotype and/or cancer induced by an oncogene whoseproduct functions upstream of a mob-5 gene product, comprisinginhibiting formation of a Mob-5 receptor in a cell containing theupstream oncogene so as to inhibit the expression of the cellulartransformation phenotype of the upstream oncogene. For example, byinhibiting formation of a Mob-5 receptor in a cell, the cellulartransformation phenotype of upstream oncogenes such as ras-oncogene, MAPkinase and raf can be inhibited.

The term Aoncogene@ refers to genes that produce products involved inaltering cellular metabolism and often stimulate unregulated growthassociated with malignant transformation of cells. Examples of oncogenesinclude but are not limited to, ras, src, myc, and fos. The cellulartransformation phenotype of this invention can be induced by a rasoncogene product.

Screening Methods

The present invention also provides a method of screening for aninhibitor of IL-22R1/IL-20R2 dimerization comprising: a) transfecting acell with a plasmid containing a nucleic acid comprising a nucleic acidsequence encoding Il-22R1 and a plasmid comprising a nucleic acidsequence encoding Il-20R2; b) contacting the cell with a putativeinhibitor and; measuring dimerization, wherein a decrease indimerization in the cell of step b) as compared to dimerization in acell that was not contacted with the putative inhibitor indicates thepresence of an inhibitor of IL-22R1/IL-20R2 dimerization.

The present invention also provides a method of screening for aninhibitor of IL-22R1/IL-20R2 dimerization comprising: a) contacting thecell comprising a nucleic acid sequence encoding IL-22R1 and a nucleicacid sequence encoding IL-20R2 with a putative inhibitor and; b)measuring dimerization, wherein a decrease in dimerization in the cellof step a) as compared to a cell comprising a nucleic acid sequenceencoding IL-22R1 and a nucleic acid sequence encoding IL-20R2 that wasnot contacted with the putative inhibitor indicates the presence of aninhibitor of IL-22R1/IL-20R2 dimerization.

The present invention further provides a method of screening for aninhibitor of EL-20R1/IL-20R2 dimerization comprising: a) transfecting acell with a plasmid containing a nucleic acid comprising a nucleic acidsequence encoding Il-20R1 and a plasmid comprising a nucleic acidsequence encoding Il-20R2; b) contacting the cell with a putativeinhibitor and; measuring dimerization, wherein a decrease indimerization in the cell of step b) as compared to dimerization in acell that was not contacted with the putative inhibitor indicates thepresence of an inhibitor of IL-20R1/IL-20R2 dimerization.

Also provided by the present invention is a method of screening for aninhibitor of IL-20R1/IL-20R2 dimerization comprising: a) contacting acell comprising a nucleic acid sequence encoding IL-20R1 and a nucleicacid sequence encoding IL-20R2 with a putative inhibitor and; b)measuring dimerization, wherein a decrease in dimerization in the cellof step a) as compared to dimerization in a cell comprising a nucleicacid sequence encoding IL-20R1 and a nucleic acid sequence encodingIL-20R2 that was not contacted with the putative inhibitor indicates thepresence of an inhibitor of IL-20R1/IL-20R2 dimerization.

An example of this screening method is a method of screening for aninhibitor of dimerization comprising: a) transfecting a cell with aplasmid containing a nucleic acid comprising a nucleic acid sequenceencoding Il-20R1 functionally linked to a flourescence donor and aplasmid comprising a nucleic acid sequence encoding IL-20R2 functionallylinked to a flourescence donor; b) contacting the cell with theinhibitor; and c) measuring fluorescence resonance energy transfer(FRET), wherein a decrease in FRET as compared to FRET measurement in acell that was not contacted with the inhibitor indicates the presence ofan inhibitor of Il-20R1/Il-20R2 dimerization.

In performing the screening methods described above, a single plasmidcan be utilized to deliver both the nucleic acid encoding IL-22R1 andthe nucleic acid encoding IL-20R2. Furthermore, either IL-22R1 orIL-20R2 can be linked to a donor or an acceptor. Similarly, a singleplasmid can be utilized to deliver both the nucleic acid encodingIL-20R1 and the nucleic acid encoding IL-20R2. Furthermore, eitherIL-20R1 or IL-20R2 can be linked to a donor or an acceptor.

For dimerization measurements, one of skill in the art could measure theamount of dimerization in a cell comprising a nucleic acid sequenceencoding IL-22R1 and a nucleic acid sequence encoding IL-20R2 before theaddition of a putative inhibitor and then measure dimerization after theaddition of the putative inhibitor. A decrease in dimerization aftercontacting the cell with the putative inhibitor is indicative of aninhibitor of IL-22R1/IL-20R2 dimerization. Alternatively, one of skillin the art could compare the amount of dimerization in cell comprising anucleic acid sequence encoding IL-22R1 and a nucleic acid sequenceencoding IL-20R2 after the addition of a putative inhibitor with theamount of dimerization in control cells comprising a nucleic acidsequence encoding IL-22R1 and a nucleic acid sequence encoding IL-20R2that have not been contacted with the putative inhibitor. Similarly, theabove dimerization measurements can be performed to determine the amountof dimerization between IL-20R1 and IL-20R2.

The present invention also contemplates a method of screening for aninhibitor of Mob-5 binding to the Mob-5 receptor comprising: contactinga Mob-5 receptor containing cell with a labeled Mob-5 protein and aputative inhibitor; measuring the amount of labeled Mob-5 protein boundto the cells, such that a decrease in labeled Mob-5 protein binding ascompared to Mob-5 protein binding in cells that were not contacted withthe inhibitor, indicates the presence of an inhibitor of the interactionbetween Mob-5 and the Mob-5 receptor. In the method described above, oneof skill in the art would compare the amount of labeled Mob-5 bound tocells that have been contacted with a putative inhibitor with the amountof Mob-5 bound to cells that were not contacted with the inhibitor. Adecrease in the amount of labeled Mob-5 bound to cells that have beencontacted with a putative inhibitor as compared to labeled Mob-5 bindingin cells that were not contacted with the inhibitor, indicates thepresence of an inhibitor of the interaction between Mob-5 and the Mob-5receptor.

The present invention also provides a method of screening for aninhibitor of Mob-activation comprising: a) contacting a Mob-5 receptorcontaining cell with Mob-5 and a putative inhibitor; and b) measuringSTAT activation, wherein a decrease in STAT activation in the cell ofstep a) as compared to a Mob-5 receptor containing cell that was notcontacted with the putative inhibitor indicates the presence of aninhibitor of Mob-5 activation.

The present invention also provides a method of screening agents foranti-cancer activity comprising; a) administering the agent to a cancercell; b) monitoring the activity of a Mob-5 receptor in the cell,whereby an inhibition of the activity of the Mob-5 receptor indicatesthe agent has anti-cancer activity, thereby screening the agent foranti-cancer activity.

Cancer Detection

The present invention also provides a method of detecting the presenceof cancer in a patient comprising: contacting a sample from the patientwith an antibody to a Mob-5 receptor as described above; detecting thebinding of the antibody with an antigen in the sample, wherein bindingof antigen to the antibody indicates the presence of Mob-5 receptorantigen in the sample and wherein Mob-5 receptor antigen in the sampleindicates the presence of cancer in the patient, thereby detecting thepresence of cancer in the patient.

In this detection method, the amount of antibody that is bound to anantigen in the sample can be compared to the amount of antibody that isbound to antigen in a sample obtained from a patient that does not havecancer. If the amount of antibody that is bound to antigen in the samplefrom the patient suspected of having cancer is greater than the amountof antibody that is bound to antigen in the sample from the patient thatdoes not have cancer, cancer is present in the patient suspected ofhaving cancer. Alternatively, one of skill in the art would know how tocompare the amount of antibody bound to antigen in the sample with theamount of Mob-5 receptor antigen detected in other cancer patients andthus, be able to determine whether or not the level of Mob-5 receptordetected corresponds to a cancerous condition.

The Asubject@ or Apatient@ of this method can be any animal. In apreferred embodiment, the animal of the present invention is a human. Inaddition, non-human animals which can be treated by the methods of thisinvention can include, but are not limited to, cats, dogs, birds,horses, cows, goats, sheep, guinea pigs, hamsters, gerbils and rabbits.

The sample of this invention can be from any organism and can be, but isnot limited to, peripheral blood, plasma, urine, saliva, gastricsecretion, feces, bone marrow specimens, primary tumors, embedded tissuesections, frozen tissue sections, cell preparations, cytologicalpreparations, exfoliate samples (e.g., sputum), fine needle aspirations,amnion cells, fresh tissue, dry tissue, and cultured cells or tissue. Itis further contemplated that the biological sample of this invention canalso be whole cells or cell organelles (e.g., nuclei). The sample can beunfixed or fixed according to standard protocols widely available in theart and can also be embedded in a suitable medium for preparation of thesample. For example, the sample can be embedded in paraffin or othersuitable medium (e.g., epoxy or acrylamide) to facilitate preparation ofthe biological specimen for the detection methods of this invention.

The methods of the present invention can be utilized to detect varioustypes of cancers including, but not limited to, pancreatic cancer, skincancer, larynx cancer, lung cancer, breast cancer, adrenocarcinoma andcolon cancer.

As used herein, “antigen” when used in the detection context generallymeans detecting the antigen, specifically the Mob-5 receptor, or afragment thereof. The antigens of this invention can also be used todetect antibodies to the Mob-5 receptor or fragments thereof. Incancerous conditions, the antigen may exist on the cell surface as wellas be detectable in a body fluid.

One example of the method of detecting the antigen is performed bycontacting a fluid or tissue sample from the patient with an amount of aan antibody, possibly purified, reactive with the antigen, cellscontaining the antigen, or fragments of the antigen, and detecting thereaction of the antibody with the antigen. The fluid sample of thismethod can comprise any body fluid which would contain the antigen or acell containing the antigen, such as blood, plasma, serum, saliva andurine, sputum, mucus and the like. An antibody used to detect theantigens of this invention is preferably specifically reactive with theantigen.

In the present invention, the step of detecting the binding of theantibody with the antigen can be further aided, in appropriateinstances, by the use of a secondary antibody or other ligand which isreactive, either specifically with a different epitope ornonspecifically with the ligand or reacted antibody. The antibody can belabeled with a detectable marker.

Enzyme immunoassays such as immunofluorescence assays (IFA), enzymelinked immunosorbent assays (ELISA) and immunoblotting can be readilyadapted to accomplish the detection of the antigen. An ELISA methodeffective for the detection of the antigen can, for example, be asfollows: (1) bind the antibody to a substrate; (2) contact the boundantibody with a fluid or tissue sample containing the antigen; (3)contact the above with a secondary antibody bound to a detectable moiety(e.g., horseradish peroxidase enzyme or alkaline phosphatase enzyme);(4) contact the above with the substrate for the enzyme; (5) contact theabove with a color reagent; (6) observe color change. Other assays fordetecting the binding of an antibody to an antigen can be used.

The present invention further provides a kit for detecting the bindingof an antibody to the Mob-5 receptor, or a fragment thereof.Particularly, the kit can detect the presence of an antigen specificallyreactive with the antibody or an immunoreactive fragment thereof. Thekit can include an antibody bound to a substrate, a secondary antibodyreactive with the antigen and a reagent for detecting a reaction of thesecondary antibody with the antigen. Such a kit can be an ELISA kit andcan comprise the substrate, primary and secondary antibodies whenappropriate, and any other necessary reagents such as detectablemoieties, enzyme substrates and color reagents as described above. Thediagnostic kit can, alternatively, be an immunoblot kit generallycomprising the components and reagents described herein. The particularreagents and other components included in the diagnostic kits of thepresent invention can be selected from those available in the art inaccord with the specific diagnostic method practiced in the kit. Suchkits can be used to detect the binding of the antibody with Mob-5receptor, or a fragment thereof, in tissue and fluid samples from apatient.

One skilled in the art will be able to correlate the levels of Mob-5receptor antigen detected using the methods disclosed herein with aparticular stage of the cancer, thus utilizing the detection method forprognostic purposes. The prognostic evaluation can determine what typeof anti-cancer therapy to employ at different stages of cancer dependingon the amounts of Mob-5 receptor antigen detected in the patient=ssample.

The invention also provides for a method of in vivo detection of Mob-5receptor by administering an anti-Mob-5 receptor antibody conjugated toa tracer to a subject and imaging the anti-Mob-5 receptor antibody.Tracers that may be conjugated to the anti-Mob-5 receptor antibody areknown in the art and include radiolabels such as 99mTc, 111In, 125O,131I. Imaging techniques are also known in the art and includeimmunoscintography, single photon emission computed tomographic imagingand high-resolution gamma-camera imaging (Sato et al. 1999.AIntratumoral distribution of radiolabeled antibody andradioimmunotherapy in experimental liver metastases model of nude mouse@J. Nucl. Med. 40:685-692; Reilly 1993 AImmunoscintography of tumoursusing 99Tcm-labelled monoclonal antibodies: a review@ Nucl. Med. Commun.14:347-359.) One skilled in the art would be able to select theappropriate combination of tracer and imaging technique to detect theanti-Mob-5 receptor antibody in vivo. The in vivo imaging of anti-Mob-5receptor antibody can be utilized for diagnostic purposes, prognosticpurposes as well as for the intraoperative detection of metastaticdeposits.

The invention also provides for a method of classifying a cancer as acolorectal cancer comprising, obtaining a sample from a patientdiagnosed with cancer, contacting the sample with an antibody to Mob-5receptor, and detecting the binding of the antibody with an antigen inthe sample, wherein the binding of the antibody to the antigen indicatedthe cancer is colorectal cancer, thereby classifying the cancer as acolorectal cancer.

By Aclassifying@ is meant to determine that the expression of Mob-5corresponds to a specific type of cancer, such as colorectal cancer andnot to another type of cancer, thus classifying the cancer. To ascertainthat the cancer is, in fact, a colorectal cancer, the detection of Mob-5receptor in the sample can be combined with the detection of anotherspecific tumor marker to confirm that the cancer is the specific type ofcancer.

By Adiagnosed@ is meant that one skilled in the art has determinedeither by blood test, immunoassay, ultrasound, urinalysis, magneticresonance imaging, physical examination, biopsy or any other diagnosticmethod that the patient has cancer.

The invention further provides a method of classifying a cancer as acolorectal cancer comprising, obtaining a sample from a patientdiagnosed with cancer, contacting the sample with a Mob-5 receptorantigen, and detecting the binding of the antigen with a Mob-5 receptorantibody in the sample, wherein the binding of the antigen to theantibody indicates the cancer is a colorectal cancer, therebyclassifying the cancer as a colorectal cancer. The detection of bindingof Mob-5 antigen to Mob-5 receptor antibody in the sample can beaccomplished by utilizing immunological detection methods previouslydiscussed.

The invention further provides a method of detecting Mob-5 receptorantibody in a sample comprising contacting a sample with a Mob-5receptor antigen, and detecting the binding of the antigen with a Mob-5receptor antibody in the sample, wherein the binding of the antigen tothe antibody indicates the presence of Mob-5 receptor antibodies in thesample.

The presence of Mob-5 receptor antibodies in a sample could be utilizedto detect autoimmune disorders such as inflammatory bowel disease,multiple sclerosis, rheumatoid arthritis, diabetes mellitus, perniciousanemia, autoimmune gastritis, psoriasis, Bechet=s disease, idiopathicthrombocytopenic purpura, Wegener=s granulomatosis, autoimmunethyroiditis, autoimmune oophoritis, bullous pemphigoid, pemphigus,polyendocrinopathies, Still=s disease, Lambert-Eaton myastheniasyndrome, myasthenia gravis, Goodposture=s syndrome, autoimmuneorchitis, autoimmune uveitis, systemic lupus erythematosus, Sjogren=ssyndrome and ankylosing spondylitis.

The present invention is more particularly described in the followingexamples which are intended as illustrative only since numerousmodifications and variations therein will be apparent to those skilledin the art.

EXAMPLES

Cell Lines and Culture

All cell lines including 293T, HaCaT and BHK (from ATCC) were grown inDulbecco=s Modified Eagle Medium (DMEM) (Life Technologies Inc. GrandIsland, N.Y.) with 10% fetal bovine serum (HyClone, Logan, Utah) and 1%penicillin-streptomycin (Life Technologies Inc. Grand Island, N.Y.) at37° C. with 10% CO₂. Cos-E5, a clonally purified Cos-1 cell line, wasmaintained in DMEM with 10% Bovine calf serum and 1%penicillin-streptomycin.

RNA Purification, RT-PCR and Southern Blot Hybridization

Total RNA from human cancer tissues or cultured cells was purified usingthe RNApure reagent following the manufacturer=s instructions (GenHunterCorp., Nashville, Tenn.). The cDNA synthesis, RT-PCR and southern blothybridization were carried out as described previously (7). The methodfor quantitative analysis of human IL-24 mRNA by competitive RT-PCR wasessentially according to the method described previously (23). First, acompetitive IL-24 cDNA template with 91 bp deletion from the native cDNAsequence was constructed by PCR with primers LhMob-4(5=-TGCAAAGCCTGTGGACTTTAGCCAGGTATCAG-3=) (SEQ ID NO: 8) and RhMob5-2(5=-CCGCCTGTGTGCACTGTCTCTGATG-3=) (SEQ ID NO: 9). The resulting 397 bpcompetitor PCR product was cloned into the PCR-TRAP cloning vector(GenHunter Corp., Nashville, Tenn.). The cloned IL-24 competitor cDNAwas purified and the same amount of an optimal concentration of thecompetitor template was included in each RT-PCR reaction using primersLhMob3 (5=-TGCAAAGCCTGTGGACTTTAGCCAG-3=) (SEQ ID NO: 12) and RhMob5-2.The signal of the 488 bp native IL-24 PCR product was separated fromthat of the 397 bp competitor by 1.5% agarose gel electrophoresis.

Production of Secreted AP, IL-24-AP Fusion Proteins and His-Tagged IL-24(IL-24-His)

Production of secreted AP and IL-24-AP fusion proteins were describedpreviously (7, 24). For the production of human IL-24-His, the PCRprimer was used to attach 9XHis to the C terminus of IL-24, and theresulting PCR product was subcloned into the Bgl II site of the pAPtag-5expression vector (GenHunter Corp., Nashville, Tenn.). Aftertransfecting into the 293T cells, stable cell lines constitutivelysecreting IL-24-His into the conditioned medium were obtained.

AP and IL-24-AP Binding and Cell Staining Assays

Receptor binding studies using AP activity and in situ cell stainingassays were carried out using AP assay reagent A and AP assay reagent S,respectively (GenHunter Corp., Nashville, Tenn.), followingmanufacture=s instruction as previously described (7).

Antibody Preparation and Immunoblotting

The polyclonal antibody to human IL-24 was described previously (7). Formonoclonal antibody production, bacterially expressed human IL-24-6XHis(7) was purified as a denatured protein. After dialysis in PBS, 25-100μg of recombinant protein in Fruend's complete adjuvant was injectedinto 4-5 week old female Balb/C mice. After three weeks, sera fromimmunized mice were assayed by ELISA for the titer of anti-IL-24antibody, using human IL-24-AP as an antigen as well as a reporter. Micewere then boosted with an additional 25-100 μg of purified humanIL-24-AP fusion protein in order to enhance the production of antibodiesrecognizing native, rather than, denatured IL-24. Three days prior tofusion, mice were hyper-immunized, again with 25-100 μg of IL-24-AP.Spleens from hyper-immunized mice were then fused with SP2/O myelomacells and hybridomas producing anti-Mob5 specific hybridomas weresubcloned according to standard procedures.

Deglycosylation

IL-24-His was partially purified from the conditioned medium usingNi-NTA beads (Qiagene). Deglycosylation of IL-24-His was carried outwith endoglycanase F (PNGase F) (Roche Molecular Chemicals) following apreviously described procedure (25).

Purification of PBMCs and Induction of IL-24 by Concanavalin A (ConA)

Human PBMCs were freshly purified from healthy donors by gradientcentrifugation using LSM lymphocyte separation medium (ICN, Costa Mesa,Calif.) according to manufacturer=s instruction. After resuspended inRPMI medium containing 10% FBS, the cells was stimulated with eitherbacterial lipopolysacharide LPS (100 ng/mL) or ConA (25 μg/mL) for 2 and4 hours. Following the treatment, the conditioned media were analyzedfor IL-24 production by western blot analysis as described (7) using amonoclonal antibody to human IL-24 (clone L14).

Transfection and Gel-Shift Assay

The coding regions of human IL-10R2, IL-20R2 and IL22-R1 were PCRamplified and subcloned between the Kpn I and Xba I sites of theexpression vector pcDEF3. The complete coding region of human IL-20R1was PCR amplified using an EST clone (IMAGE # 2700520) (ATCC, Manassas,Va.) as a template and subcloned into the Bgl II site of the pAPTag-5expression vector. Expression plasmids encoding different receptorsubunits were transiently transfected into either Cos-E5 or BHK cellsusing FUGENE-6 (Roche Molecular Chemicals) according to manufacturer=sinstruction. Two days after transfection, cells were treated with 1:10dilution (directly into the existing culture media) of the 293Tconditioned medium either without or with IL-24-His (final concentrationat about 50 ng/mL). Thirty minutes following the treatment, nuclearextracts were prepared by the standard protocol. For STAT specificgel-shift assays, STAT-specific double stranded DNA probe GRR(5=-ATGTATTTCCCAGAAA-3=(SEQ ID NO: 10)/5=-CCTTTTCTGGGAAATAC (SEQ IDNO:11)-3=) was end labeled with ³²P, incubated with nuclear extracts andseparated on 5-6% non-denaturing polyacrylamide gels as describedpreviously (3). For super-shift experiments, 1 μg of STAT-1 or STATB3specific antibody was used (Santa Cruz).

LPS Induced TNF-α Production from PBMCs

Freshly prepared PBMCs (see above) were resuspended in RPMI plus 10% FBSand equal aliquots (150 μL) were plated in a 96-well plate. LPS was thenadded to each well at a final concentration of 80 ng/mL in the absenceor presence of either 20 ng/mL of recombinant human IL-10 (R&D Systems,Minneapolis, Minn.) or 100 ng/mL of human IL-24-His, or both for fivehours. TNF-α levels in the conditioned media of the cells weredetermined with an ELISA kit for human TNF-α (R&D Systems, Minneapolis,Minn.) as instructed by the manufacturer.

Overexpression of IL-24 in Human Colon Cancer

To determine the relevance of IL-24 expression in human cancer is ofgreat importance for the present invention=s model system forras-mediated cell transformation (7). To evaluate the expression ofIL-24 in colorectal cancer which has frequent mutations in the rasproto-oncogene, quantitative RT-PCR analysis with RNA isolated from thetumors and the adjacent normal tissues from five colorectal cancerpatients was carried out (FIG. 1A, upper panel). The quantitative RT-PCRresults were also confirmed by regular RT-PCR of the entire codingregion of IL-20 followed by Southern blot using human IL-24 cDNA as aprobe (FIG. 1A, middle panel). These results showed that IL-24expression was confined to cancer tissues from five out five patients,with patient No. 5 expressing IL-24 at a much higher level.

Induction of IL-24 by Activated T cells

Although IL-24 expression in rodents was not detectable in any normaladult tissues or in embryos (7), it was reported by others that the geneappeared to be induced under pathological conditions such as woundhealing (6). Given the fact that IL-24 belongs to the IL-10 family ofcytokines which can be induced by ConA activated T cells (11, 25), IL-24induction under such conditions was analyzed. Freshly prepared humanPBMCs were stimulated with either bacterial LPS or Con A. Followingstimulation, the production of IL-24 in the conditioned media wasanalyzed by Western blot (FIG. 1B). ConA, which causes mostly T cellactivation, induced a rapid secretion of IL-24 by the PMNCs. LPS, on theother hand, had little effect on IL-24 production.

IL-24 is a Glycosylated Protein

The predicted molecular weight of human IL-24 is 23.8 kDa, but the IL-24secreted by either human PBMCs (FIG. 1B) or IL-24-His secreted by 293Tstable transfectants appeared to be much bigger with a molecular weightcloser to 35 kDa (FIG. 2). To determine whether this is due toglycosylation, partially purified IL-24-His was subjected to digestionby endoglycanase F (PNGase F). As predicted, the deglycosylating enzymeconverted the 35 kDa form of IL-24-His to several smaller molecularweight forms, with the lowest band being close to 23 kDa (FIG. 2B). Thepresence of bands of intermediate sizes could be due to an incompletedeglycosylation. This result suggests that IL-24 is extensivelyglycosylated.

Identification of the IL-24 Receptors

Since IL-24 shares significant homology to IL-10, it was predicted thatIL-24 is a member of the IL-10 family of cytokines (7). Supporting thishypothesis is the finding of this group that not only IL-24, but alsoits putative cell surface receptor appears to be induced by rasoncogenes (7). Shortly after the initial description of IL-24, two moreIL-10 family of cytokines, IL-20 and IL-22, and their receptors werereported (9-10). Sequence alignment indicated that IL-24 and the othermembers of the IL-10 family of cytokines share an overall homologyranging from 24-33% among each other. Among them, IL-24 is closest toIL-20 with 33% homolgy overall, and the highest homology is found at theC-termini of the proteins.

Based on published data and database searches of GenBank (NCBI), onlythree R1 and two R2 types of the IL-10 family of receptor subunits canbe identified. The three R1 subunits are IL-10R1, IL-20R1 and IL-22R1,whereas the two R2 subunits are IL-20R2 and IL-10R2, the latter of whichis also the second receptor subunit for IL-22. Thus, the IL-24 receptorcould be a heterodimer of a combination of the known R1 and R2receptors. To determine if IL-24 is the ligand for any of theheterodimers of known R1 and R2 receptors, receptor binding assays werecarried out using either secreted human placental alkaline phosphatase(AP) or the IL-24-AP fusion protein as a probe (FIG. 3A; 7). Thequantitative cell surface binding assays were carried out with Cos cellsafter transfecting plasmids over-expressing IL-10R2, IL-20R1, IL-22R1and IL-20R2 individually or in all four R1/R2 combinations (FIG. 3B).IL-24-AP, but not AP, exhibited significant binding to Cos cellstransfected with IL-20R2 alone, and the binding was further dramaticallyincreased when IL-20R2 was co-transfected with either IL20-R1 orIL-22R1. Neither IL20-R1 nor IL-22R1 alone was able to bind to IL-24-AP.Using AP staining assays, the quantitative IL-24-AP binding data wasconfirmed (FIG. 3C). Specific cell surface staining was detected withIL-24-AP when Cos cells were transiently transfected with IL-20R2, butnot IL-20R1 or IL-22R1 alone (FIG. 3C). When cells were transientlytransfected with IL-20R2 in combination with either IL-20R1 or IL-22R1,the cell surface binding of IL-24-AP was greatly increased (FIG. 3C). Inaddition to increased ability to bind IL-24-AP, the presence of eitherR1 subunit resulted in a much more uniformed distribution of theheterodimeric receptor over the entire cell surface (FIG. 3C).

IL-24 Signals through both the IL-20 Receptor (IL20R1/IL-20R2) andIL22R1/IL-20R2 Heterodimers

To functionally determine if IL-24 can signal through these putativereceptors by activating the downstream JAK-STAT pathways, as has beenobserved for other members of the IL-10 family of cytokines, thefollowing three experiments were performed. First, the expression ofIL-22R1 in a human keratinocyte cell line, HaCaT, which was shown to bepositive for IL-20 receptor (IL20R1/IL-20R2) was examined (Blumberg, etal., 2001). The finding of the co-expression of IL-22R1 and IL-20R2 inthe HaCaT, but not BHK cell line (FIG. 4A), suggests that the formercould contain both receptors that are capable of binding IL-24. Thegel-shift assays were then conducted for the functional analysis of STATactivation in the HaCaT cells by IL-24-His (FIG. 4B). After 30 minstimulation, IL-24-His treated cells showed a marked increase in STATactivation (FIG. 4B). It was possible that the IL-24 induced STATactivation in HaCaT cells could be due to IL-24 binding to either theIL-20 receptor or to IL-22R1/IL-20R2. To differentiate between these twopossibilities, vectors expressing IL-20R1 and IL20R2, or IL-22R1 andIL-20R2 were transfected into the BHK cell line, which does not haveendogenous IL-24 receptors as predicted by the lack of IL-20R2expression. Interestingly, upon stimulation of the transfected cellswith either the control- or IL-24-His-conditioned medium, the gel shiftassay revealed that both the IL-20 receptor (IL20R1/IL-20R2) andIL-22R1/IL-20R2 heterodimeric receptor were able to confer comparableand robust IL-24 dependent STAT activation (FIG. 5A). None of thereceptor subunits alone, including IL-20R2, was able to confer IL-24dependent STAT activation. These data are consistent with the receptorbinding assays (FIG. 3). Finally, to determine which STAT was activatedby IL-24 in BHK cells transfected with the IL-24 receptor(IL-22R1/IL-20R2), antibodies against different STATs were used tointerfere with the gel-shift assays. A higher resolution band-shift gelshowed that IL-24 binding to its heterodimeric receptor led to theactivation of both STAT-1 and B3, which could be interfered (for STAT-1)or super-shifted (for STAT-3) by the corresponding antibodies,respectively (FIG. 5B).

Effect of IL-24 on LPS Induced TNF-α Production

IL-10 is known to be a potent inhibitor of cytokine synthesis induced byLPS (27). To determine if IL-24 has a similar biological activity,freshly prepared human PBMCs were stimulated with LPS in the absence orpresence of IL-10, IL-24 or both. The effects of IL-10 and IL-24 on LPSinduced TNF-α production were determined by ELISA. Unlike IL-10, IL-24did not appear to have any inhibitory effect on TNF-α induction by PBMCs(FIG. 5).

Induction of One of the IL-24 Receptors, IL-20 Receptor, by Ha-rasOncogene

Previous results showed ras oncogenes can activate not only IL-24, butalso its cell surface receptor(s) (Zhang et al., 2000). An EST data basesearch revealed several cDNA matches to both rodent IL-20R1 and IL-20R2,but not IL-22R1, Using these cDNAs as probes, we showed that compared tothe parental primary rat embryo fibroblasts (REF), REF transformed byoncogenic Ha-ras and mutant p53 (Martinez, 1991), had overexpression ofnot only IL-24, but also IL-20 receptor (IL-20R1/IL-20R2, which is alsothe second IL-24 receptor) (FIG. 7). This result shows that rasoncogenes can activate IL-24/IL-24Rs autocrine loops. It should be notedthat Rat-1 cells transformed by Ha-Ras also had an elevated expressionof IL-20 receptor, which is lower, however, than that found in T101-4and A1-5 cells. It is possible that the inactivation of p53 couldfurther enhance the ras activation of IL-24/IL-24R autocrine loop. Byutilizing the rodent cDNA for IL-22R1, one of skill in the art candetermine if the other IL-24 receptor (IL-22R1/IL20R20) is alsoactivated by ras oncogenes.

When treated with ha-RAS inhibitor FTI, T101-4 cells showed rapiddecrease in IL-20R2 expression. In contrast, MAPK kinase inhibitor,PD98059 had less effect. This result re-enforces that ras oncogenes,through multiple pathways, can activate an IL-24/IL-24R autocrine loop.It should be noted that Rat-1 cells transformed by Ha-Ras also had anelavated expression of IL-20 receptor which is lower, however, than thatfound in T101-4 and A1-5 cells It is possible that the inactivation ofp53 could further enhance the ras activation of IL-24/IL-24R autocrineloop.

IL-24 Signaling Pathway Can Support Cell Survival and Proliferation

In order to understand the functions of IL-24 with a clear readout incell survival and proliferation, an IL-3 dependent murine pro-B cellline, Ba/F3 was utilized (Rodriguez-Tarduchy et al. 1990 (30), 1990;Devireddy et al., 2001 (31)). This cell line depends on IL-3, inaddition to fetal bovine serum, for survival and growth. Upon IL-3withdraw, Ba/F3 cells undergo rapid apoptosis within 24 hours(Rodriguez-Tarduchy et al., 1990 (30)).

After stably expressing either of the IL-24 receptors in Ba/F3 cells(pooled G418 resistant clones), which were confirmed by ligand binding,it was determined whether or not IL-24 could substitute IL-3 insupporting cell viability and growth. Using a cell viability assay withAlama Blue dye (Liu et al., 1994), IL-24 was shown to be able to preventcell death in an IL-24 receptor-specific manner. The result was alsoconfirmed by apoptosis assay for chromosomal DNA fragmentation asreported previously (Rodriguez-Tarduchy et al., 1990). Because the cellviability assay could not tell living cells from growing ones, IL-24dependent cell growth was measured. Ba/F3 cells transfected with vectoralone were obligatorily IL-3 dependent whereas Ba/F3 cells transfectedwith the either of the IL-24 receptors could depend on either IL-3 andIL-24 for long term proliferation.

These functional results for IL-24 provide strong evidence that an IL-24autocrine (or paracrine) loop plays a key role in mediating a rasoncogene effect in supporting both tumor cell survival andproliferation.

Colon Cancer Cell Line, SW480, is Positive for IL-24 Receptor(s)

To obtain more evidence for the presence of IL-24 receptor in humancancer cells, a colon cancer cell lines SW480, which was shown tocontain both mutations in Ka-ras and p53 was examined. Stimulation ofthe cells with recombinant IL-24 led to increase in STAT activation asdetermined by gel-shift assay (FIG. 8).

The identification of two functional IL-24 receptors, one consisting oftwo recently identified class II cytokine receptors, IL-22R1 andIL-20R2, the other being the IL-20 receptor (IL-20R1/IL-20R2), showsthat IL-24 is indeed a new member of IL-10 family of cytokines,following IL-20 and IL-22. The observation that Mob-5-AP exhibitednearly identical properties to that of human IL-24-AP in receptorbinding studies presented in FIG. 3, shows that Mob-5 and Mda-7 are therat and human homologs of IL-24, respectively.

The three previously known receptors of the IL-10 family of cytokinesare all heterodimeric, each being made up of a R1 type and a R2 type ofreceptor subunit, IL-10 and IL-22 share the same R2 type of receptorsubunit, IL-10R2. This invention shows that IL-24 not only shares thesame R2 type of receptor subunit with that of IL-20, but also its R1type of receptor subunit is shared with that of IL-22. The discovery ofsuch a scheme of receptor subunit swapping is important in two aspects.First, there could be a total of six combinations of heterodimericreceptors made up of three known R1 and two known R2 receptor subunits,to which four combinations now have already assigned ligands, IL10,IL-20, IL-22 and IL-24 (Table I). Thus, two more receptors made up ofIL10R1/IL20R2 and IL-20R1/IL10R2 have yet to be assigned with theircorresponding ligands. These may correspond with two more IL-10 likecytokines that do now have known receptors, IL-19 (8) and AK155 (4),both of which are encoded by cellular genes. The receptor-ligandrelationship presented in this invention will be helpful for theidentification and assignment of other receptors and their correspondingligands.

Second, the scheme of receptor subunit swapping of the present inventionshows that there is crosstalk among IL-10 family of cytokines (10).Clearly shown by this study, both IL-20 and IL-24 can signal through thesame IL-20 receptor. Conversely, IL-20 may also be able to signalthrough the other new IL-24 receptor (IL-22R1/IL-20R2). Moreover, thefinding that IL-22R1 is the first R1 type of receptor subunit shared bytwo different ligands of the IL-10 family of cytokines, would alsopredict a potential crosstalk between IL-22 and IL-24. For example,although the studies presented herein showed that IL-24 could not signalthrough the IL-22 receptor (IL-22R1/IL-10R2), IL-22 may be able tosignal through the IL-24 receptor(s). Such potential crosstalk amongIL-10 family of cytokines and their receptors can now be readily testedas a result of the present invention.

Dysfunction in the control of such ligand-receptor crosstalk as a resultof aberrant expression of the cytokines and their receptors could be oneof the underlying causes for diseases, such as cancer. The presentinvention shows that ras oncogenes cause constitutive expression ofIL-24 in cells from non-hematopoietic origin (7). Ras transformed ratembryo fibroblasts and intestinal epithelial cells not onlyconstitutively produce IL-24, but also have increased expression of theIL-24 receptor(s) (7). Like IL-20, one of the likely normal targettissues for IL-24 is skin, since the human keratinocyte cell line HaCaTappears to express both endogenous IL-24 receptors. This is alsoconsistent with the finding that IL-24 is transiently expressed inlesions of skin injury from a rat wound-healing model (6). It should bepointed out that the human IL-24 gene (mda-7), when ectopicallyexpressed, was reported to have marginal growth inhibitory effect onsome cancer cell lines (29), which is in paradox with our finding thatthe gene is both induced by ras oncogene and overexpressed in coloncancer tissues. While little expression of IL-22R1 was detected fromcolon and lung, over-expression of the gene was found in cancer celllines from these tissue origins (10). Virally encoded IL-10 likecytokines may participate in cell infection and transformation, and actthrough Il-24 receptor(s).

Throughout this application, various publications are referenced. Thedisclosures of these publications in their entireties, as well as thereferences cited in these publications, are hereby incorporated byreference into this application in order to more fully describe thestate of the art to which this invention pertains.

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1-6. (canceled)
 7. A purified antibody that specifically binds to acomposition comprising a complex between IL-22R1 and IL-20R2. 8-48.(canceled)